Aging tendons become more susceptible to injury and less able to heal damage. Ultimately, declines in tendon function with age must reflect changes in the tenocytes that maintain the tissue, and in the tissue-resident stem cells that replenish the tenocyte population throughout adult life. Declines in overall tendon cell numbers with age have been reported, suggesting that a progressive loss in the ability to replenish the tenocyte population may contribute to impaired tissue function. The goals of this proposal are i) to define age-related changes in human tendon tissue-resident stem cells, ii) to identify mechanisms responsible for stem cell maintenance, and iii) to test whether those mechanisms can be manipulated to enhance tendon repair. In preliminary studies, we found that the frequency of Tendon Stem/Progenitor Cells, or TSPCs is markedly reduced in aged vs. young tendons of rats and humans, consistent with reduced overall tendon cellularity. Furthermore, TSPCs from aged tendons proliferate slowly and show evidence of cell cycle arrest in the G2 phase. Our preliminary study indicates that the transcription factor CITED2 (CREB-binding protein/p300-interacting transactivator with ED- rich tail), a cell growth regulator and potent suppressor of cel senescence, is strongly downregulated in aged TSPCs, and its knockdown by shRNA increases cell cycle arrest. Our pilot data also indicate that CITED2 regulates several genes governing adult stem cell self-renewal. Based on these findings (Aim 1), we will determine TSPC content and functionality (proliferation rate, cell cycle status, differentiation potential) in human tendos at different ages. Next (Aim 2), we will determine the role of CITED2 in TSPC maintenance with age using gain and loss of function approaches to determine the role of CITED2 in TSPC proliferation, cell cycle progression and apoptosis. We will also assess the ability of CITED2 to regulate key steps in pathways controlling cell proliferation and survival. Lastly (Aim 3), we will determine the ability of CITED2 to enhance tendon repair by TSPCs in vivo. We will compare the ability of TSPCs from young and old tendons to repair patellar tendon damage in vivo, then test whether overexpression of CITED2 in these cells will improve cell repopulation and healing. We will carry out similar experiments using bone marrow-derived stem cells (MSCs) isolated from the same donors, as MSCs are considered an excellent source of stem cells for regeneration of tendons and other tissues. These studies will identify functional changes that occur in human TSPCs with age (Aim 1), establish whether a proposed novel mechanism involving CITED2 can account for age-related changes in human TSPC function (Aim 2), and test whether manipulation of CITED2 can enhance the ability of stem cells to repair tendon damage in vivo (Aim 3). These findings may provide new insights into the basis for age-related tendon disorders, and the development of new strategies for tendon repair and regeneration.